PROJECT SUMMARY The ability of synapses to change their functional and structural properties in response to activity, called synaptic plasticity, is believed to act as the molecular basis of learning and memory and relies critically on protein synthesis. The regulation of protein synthesis is essential for normal synaptic function and is altered in synaptopathic diseases like Fragile X Syndrome (FXS) and Autism Spectrum Disorders (ASDs). Despite years of research, these diseases still have no effective treatment and place a large burden on society, since many adults with FXS/ASDs require a caregiver and cannot hold a job. Synaptic plasticity can occur as a result of postsynaptic receptor modifications or presynaptic changes in neurotransmitter release. While most studies of FXS have focused on postsynaptic plasticity mechanisms, including local protein synthesis, relatively little is known about the mechanisms underlying long-term presynaptic changes in the context of FXS. The Fragile X mental retardation protein (FMRP), mutated in FXS, is an mRNA binding protein that regulates activity-dependent local protein synthesis. FMRP is expressed presynaptically, but its role in long-term presynaptic plasticity has never been investigated. Utilizing cutting-edge imaging techniques like super- resolution microscopy, and two-photon laser scanning microscopy (2PLM) coupled with electrophysiology, this proposal will investigate presynaptic protein synthesis, and its regulation by FMRP, in activity-dependent plasticity. Fluorescent Noncanonical Amino Acid Tagging (FUNCAT) will be used to measure local presynaptic protein synthesis in the context of presynaptic plasticity. Presynaptic manipulations of protein synthesis will enable investigation of the cell-specific mechanisms that lead to enduring changes in neurotransmitter release at the presynapse. To test the role of presynaptic FMRP, a conditional knock out model of FMRP will be used to specifically delete the protein in the presynaptic cell. Two-photon microscopy and electrophysiology will enable real-time assessment of the mechanisms that govern activity-dependent presynaptic structural and functional plasticity. This proposal will uncover heretofore unknown mechanisms of long-term changes in neurotransmitter release, a previously neglected line of research, and likely generate new insights into the synaptic pathology of diseases like FXS.